| Rapeseed meal is a good source of plant protein with reasonable amino acid composition.In addition,it also contains functional components such as functional polysaccharides,functional peptides,phenols and lecithin.However,glucosinolates(GLs),as the most important anti-nutritional factor,limit the wide application of rapeseed meal in food.GLs can be hydrolyzed to form thiocyanates(TCs),isothiocyanates(ITCs)and nitriles(RCN)under the catalysis of myrosinase(MYR).Most of these hydrolysates have various biological activities,such as anti-cancer,anti-pathogen,anti-inflammatory,promoting auxin homeostasis,enhancing resistance,and also participate in plant defense system.In this study,LC-MS/MS technology was used to establish a quantitative method for GLs in rapeseed meal.With this method,the effects of different temperature,time,solid-liquid ratio and p H value on degradation of GLS by endogenous MYR in rapeseed meal to produce ITCs and OZT were investigated,and the optimal incubation conditions were optimized.Finally,the treatment of MYR from rapeseed sprouts for GLs degradation in rapeseed meal was studied,and the optimal hydrolysis conditions were obtained.The main results are as follows:1.Eleven kinds of GLs were detected in rapeseed meal,and their primary and secondary mass parameters were obtained,which were progoitrin,gluconapin,glucobrassicanapin,glucoraphanin,sinigrin,gluconasturtiin,gluconapoleiferin,neoglucobrassicin,4-methoxyglucobrassicin,glucobrassicin and 4-hydroxyglucobrassicin.And the first six of them were the dominant GLs in rapeseed meal.2.A method based on LC-MS/MS for quantitative determination of GLs in rapeseed meal was established,including extraction and concentration of GLs,determination of instrument technical parameters such as ion source temperature,collisionally activated dissociation and declustering potential,etc.In addition,characteristic ion pairs and collision pressure values for each GLs were obtained,and the standard working curves of six main GLs were drawn.This quantitative method was evaluated comprehensively,the results showed that the correlation coefficients R~2of the six standard curves were all above 0.9999,the recoveries were 92.44%～102.89%,the RSD of repeatability test and precision test was less than 4.14%and 3.63%,respectively.Thus,the method is accurate and reliable.3.For the degradation of GLs by endogenous MYR in rapeseed meal,the effects of different incubation conditions on the content of ITCs and OZT were studied.Temperature,time,solid-liquid ratio and p H all significantly affected the formation of ITCs and OZT.In addition,the result of orthogonal experiment showed that the optimal incubation condition was incubation time 1.5 h,incubation temperature 50°C,ratio of material to liquid 1:10and p H 7.0.Under this condition,the contents of ITCs and OZT were 4.46 and 5.25 times higher than those of non incubated rapeseed meal,respectively.The total content of six main GLs was reduced to 10.22μmol/g,and the degradation rate was 12.10%.4.The effect of MYR from rapeseed sprouts on the hydrolysis of GLs in rapeseed meal were explored.Firstly,the change of MYR activity during germination was studied.The effects of MYR dosage,hydrolysis time,AA dosage and EDTA dosage on the hydrolysis rate of GLs in rapeseed meal were also studied,and the optimal hydrolysis conditions was obtained by response surface methodology.The results showed that the activity of MYR extract was the highest and reached 2484.83 U/g(FW)after 3 days of germination followed by precipitation of ammonium sulfate with 20%～60%saturation.The optimum condition for hydrolyzing GLs in rapeseed meal was as follows:solid-liquid ratio 1:10,p H7.0,temperature 50°C,time 4 h,AA dosage 9.63μg/g and EDTA dosage26.68μg/g.Under this condition,the degradation rate of the six main GLs was 80.19%,and the yield of ITCs and OZT was 79.86 and 87.89 times higher than that of rapeseed meal,respectively.Using this method,only 11.11 g rapeseed was needed for 1 kg rapeseed meal. |
